Localization and regulation of metabolic gene expression in response to dietary triglycerides

翻译后摘要：膳食高脂肪和胆固醇的代谢反应需要一个复杂的协调 细胞过程的系统。这些细胞过程的破坏可导致代谢疾病，例如 糖尿病、心脏病和肝脂肪变性。先前的实验表明，高脂肪饮食会增加 调节脂质代谢、储存和分泌的基因和基因途径的转录。之一 在消化器官（肝脏和小肠）中，大多数高度上调的基因， 甘油三酯（TG）是载脂蛋白A4（apoa 4）。ApoA 4蛋白的功能目前尚不清楚，但它具有 涉及脂蛋白颗粒合成和分泌的调节，以及饱腹感，炎症， 和胰岛素反应性。由于TG诱导的apoa 4基因表达在培养的细胞中没有观察到， 只在整个动物模型中观察到，我将使用斑马鱼系统来检查空间和时间 脂质代谢基因的表达对膳食TG的响应。斑马鱼的消化系统在功能上 在发育上类似于哺乳动物系统，在基本代谢方面具有高度的遗传保守性， 件.因为斑马鱼幼鱼的视觉清晰，基因也容易控制，我设计了一个细胞- 追踪内源性apoa 4基因座的荧光报告基因，这将使我能够可视化饮食中的TG 在活的斑马鱼幼虫中的反应。我计划描述apoa 4表达的时间和位置，以确定 脂质代谢基因的表达是否是对TG的直接细胞摄取的细胞自主反应;或 如果它是由短距离和/或长距离细胞间信号传导驱动。 所提出的apoa 4报告基因也将使我能够筛选特定途径基因的作用， 转录因子（TF）对TG诱导的肠上皮细胞转录反应的影响， apoA 4表达。我计划用我的apoa 4报告作为衡量饮食中TG吸收的指标，以帮助描述 饮食TG代谢基因反应的细胞机制。不过，有几个TF 已经在肝脏中鉴定了调节apoa 4的TF，在肠上皮细胞中负责调节apoa 4的TF还没有 被定性。我们知道消化器官中脂蛋白的合成和分泌在很大程度上依赖于 对微粒体甘油三酯转移蛋白（MTP）活性的影响，但依赖于 不同消化器官中apoa 4基因表达的MTP未知。确定监管 为了研究肠道中MTP依赖性的潜在机制，我计划使用apoa 4转基因报告基因来检测 可能介导膳食TG吸收、MTP活性和apoa 4之间关系的基因 表情这项拟议中的研究将确定代谢基因反应之间的调控关系， 饮食TG和脂蛋白的产生和分泌。此外，所提出的apoa 4光学报告器将 是一种快速测量甘油三酯吸收的有价值的工具， 行为，微生物，药理学和环境条件在未来的研究。

Abstract: The metabolic response to meals high in fat and cholesterol requires the coordination of a complex system of cellular processes. Disruption of these cellular processes can lead to metabolic diseases, such as diabetes, heart disease, and hepatic steatosis. Previous experiments have shown that high-fat diets increase transcription of genes and gene pathways that mediate lipid metabolism, storage, and secretion. One of the most highly upregulated genes within the digestive organs (liver and small intestine), in response to dietary triglycerides (TG), is apolipoprotein A4 (apoa4). The function of ApoA4 protein is currently unknown, but it has been implicated in the regulation of lipoprotein particle synthesis and secretion, as well as satiety, inflammation, and insulin responsivity. Since TG-induced apoa4 gene expression has not been observed in cultured cells and only observed in whole animal models, I will use the zebrafish system to examine the spatial and temporal expression of lipid metabolic genes in response to dietary TG. The digestive system of zebrafish is functionally and developmentally similar to mammalian systems, with high genetic conservation in essential metabolic components. Because larval zebrafish are optically clear and genetically tractable, I have engineered a cell- tracing, fluorescent reporter in the endogenous apoa4 locus, which will allow me to visualize the dietary TG response in live zebrafish larvae. I plan to characterize the timing and location of apoa4 expression to determine whether expression of lipid metabolic genes is a cell-autonomous response to direct, cellular uptake of TGs; or if it is driven by short- and/or long-distance intercellular signaling. The proposed apoa4 reporter will also allow me to screen for the effect of specific pathway genes and transcription factors (TFs) on the TG-induced transcriptional response of intestinal enterocytes that results in apoa4 expression. I plan to use my apoa4 reporter as a measure of dietary TG absorption to help delineate the cellular mechanisms that underlie the dietary TG metabolic gene response. Although, several TFs implicated in regulating apoa4 have been identified in the liver, TFs responsible for regulating apoa4 in enterocytes have not been characterized. We know that lipoprotein synthesis and secretion in digestive organs is largely dependent on microsomal triglyceride transfer protein (MTP) activity, but the mechanisms underlying the dependence on MTP for apoa4 gene expression in different digestive organs is unknown. To determine the regulatory mechanisms underlying MTP-dependence in the intestine, I plan to use the apoa4 transgenic reporter to assay genes that potentially mediate the relationships between dietary TG absorption, MTP activity, and apoa4 expression. The proposed research will identify regulatory relationships between the metabolic gene response to dietary TGs, and lipoprotein production and secretion. Additionally, the proposed apoa4 optical reporter will be a valuable tool for rapid measurement of triglyceride absorption in response to changes in nutritional, behavioral, microbial, pharmacological, and environmental conditions in future studies.